Pharmacological characterization of a novel antiglaucoma agent, Bimatoprost (AGN 192024).

Replacement of the carboxylic acid group of prostaglandin (PG) F(2alpha) with a nonacidic moiety, such as hydroxyl, methoxy, or amido, results in compounds with unique pharmacology. Bimatoprost (AGN 192024) is also a pharmacologically novel PGF(2alpha) analog, where the carboxylic acid is replaced by a neutral ethylamide substituent. Bimatoprost potently contracted the feline lung parenchymal preparation (EC(50) value of 35-55 nM) but exhibited no meaningful activity in a variety of PG-sensitive tissue and cell preparations. Its activity seemed unrelated to FP receptor stimulation according to the following evidence. 1) Bimatoprost exhibited no meaningful activity in tissues and cells containing functional FP receptors. 2) Bimatoprost activity in the cat lung parenchyma is not species-specific because its potent activity in this preparation could not be reproduced in cells stably expressing the feline FP receptor. 3) Radioligand binding studies using feline and human recombinant FP receptors exhibited minimal competition versus [(3)H]17-phenyl PGF(2a) for Bimatoprost. 4) Bimatoprost pretreatment did not attenuate PGF(2alpha)-induced Ca(2+) signals in Swiss 3T3 cells. 5) Regional differences were apparent for Bimatoprost but not FP agonist effects in the cat lung. Bimatoprost reduced intraocular pressure in ocular normotensive and hypertensive monkeys over a 0.001 to 0.1% dose range. A single-dose and multiple-dose ocular distribution/metabolism studies using [(3)H]Bimatoprost (0.1%) were performed. Within the globe, bimatoprost concentrations were 10- to 100-fold higher in anterior segment tissues compared with the aqueous humor. Bimatoprost was overwhelmingly the predominant molecular species identified at all time points in ocular tissues, indicating that the intact molecule reduces intraocular pressure.

Replacement of the carboxylic acid group of prostaglandin f(2alpha) with a hydroxyl or methoxy substituent provides biologically unique compounds.

Replacement of the carboxylic acid group of PGF(2alpha) with the non-acidic substituents hydroxyl (-OH) or methoxy (-OCH(3)) resulted in an unexpected activity profile. Although PGF(2alpha) 1-OH and PGF(2alpha) 1-OCH(3) exhibited potent contractile effects similar to 17-phenyl PGF(2alpha) in the cat lung parenchymal preparation, they were approximately 1000 times less potent than 17-phenyl PGF(2alpha) in stimulating recombinant feline and human FP receptors. In human dermal fibroblasts and Swiss 3T3 cells PGF(2alpha) 1-OH and PGF(2alpha) 1-OCH(3) produced no Ca(2+) signal until a 1 microM concentration was exceeded. Pretreatment of Swiss 3T3 cells with either 1 microM PGF(2alpha) 1-OH or PGF(2alpha) 1-OCH(3) did not attenuate Ca(2+) signal responses produced by PGF(2alpha) or fluprostenol. In the rat uterus, PGF(2alpha) 1-OH was about two orders of magnitude less potent than 17-phenyl PGF(2alpha) whereas PGF(2alpha) 1-OCH(3) produced only a minimal effect. Radioligand binding studies on cat lung parenchymal plasma membrane preparations suggested that the cat lung parenchyma does not contain a homogeneous population of receptors that equally respond to PGF(2alpha)1-OH, PGF(2alpha)1-OCH(3), and classical FP receptor agonists. Studies on smooth muscle preparations and cells containing DP, EP(1), EP(2), EP(3), EP(4), IP, and TP receptors indicated that the activity of PGF(2alpha) 1-OH and PGF(2alpha) 1-OCH(3) could not be ascribed to interaction with these receptors. The potent effects of PGF(2alpha) 1-OH and PGF(2alpha) 1-OCH(3) on the cat lung parenchyma are difficult to describe in terms of interaction with the FP or any other known prostanoid receptor.

A single amino-acid substitution in the EP2 prostaglandin receptor confers responsiveness to prostacyclin analogs.

A high degree of homology between the four Gs-coupled prostaglandin (PG) receptors [EP2, EP4, prostacyclin (IP), PGD2 (DP)] and the four Gq/Gi-coupled receptors [EP1, EP3, PGF2alpha (FP), thromboxane A2 (TP)] suggests that prostaglandin receptors evolved functionally from an ancestral EP receptor before the development of distinct binding epitopes. If so, ligand selectivity should be determined by a limited number of amino acids. EP2 receptor transmembrane domain residues that are similar to those in the EP4 receptor but differ from those in the IP receptor were mutated to the corresponding IP receptor residue. Activation of the mutant receptors by PGE2 (EP2 ligand), iloprost (stable prostacyclin analog), and PGE1 (EP2/IP ligand) was determined using a cAMP-dependent reporter gene assay. A Leu304-to-tyrosine substitution in the seventh transmembrane domain enhanced iloprost potency approximately 100-fold. A glycine substitution at Ser120 in the third transmembrane domain had no effect on drug potency but improved the response of the Tyr304 mutant. The potency of the natural prostaglandins PGF2alpha and PGD2 was not enhanced by the mutations. In contrast, the potency of all prostaglandins was reduced 10- to 100-fold when arginine 302, which is thought to be a counterion for the prostaglandin carboxylic acid, was mutated. Thus, a single amino acid change resulted in a selective gain of function for iloprost, which is consistent with the proposed phylogeny of the prostaglandin receptors.

Muscarinic receptor subtypes in human iris-ciliary body measured by immunoprecipitation.

PURPOSE: To determine the relative levels of the five muscarinic receptor subtypes in the anterior segment of the human eye. METHODS: Antisera selective for each of the five muscarinic receptor proteins were incubated with [3H]-QNB bound receptors solubilized from human iris sphincter, ciliary muscle, and ciliary processes. Precipitation of the radiolabeled receptor-antibody complexes and scintillation counting enabled quantitation of the subtypes in the various tissues. Reverse transcription-polymerase chain reaction was performed on the tissues and cultured smooth muscle cells derived from them. RESULTS: Approximately 60% to 75% of the muscarinic receptors in the human iris sphincter and ciliary body are the m3 subtype. Lower levels (5% to 10%) of the m2 and m4 receptors are present in these tissues. The m1 receptor (7%) was detected in the ciliary processes and iris sphincter and the m5 receptor (5%), which is usually found only in the central nervous system, was present in the iris sphincter. CONCLUSIONS: The m3 subtype is the predominant muscarinic receptor in the anterior segment of the human eye. The extensive heterogeneity of muscarinic receptors makes it difficult to predict whether subtype-selective drugs will have an improved efficacy and side-effect profile.